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Author Topic: Advantages of citrate buffer?  (Read 13240 times)

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Offline doc-L

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Advantages of citrate buffer?
« on: October 05, 2004, 02:57:51 PM »
Greetings,

for my doctorate I use the method of immunohistochemistry. Since I am a newcomer to this method my question is of a more basic nature:

Why do I use a citrate buffer for antigen retrieval?
Why not TBS in this particular case?
What are the advantages of citrate buffer compared to TBS?

Thank you for answering!

Advantages of citrate buffer?
« on: October 05, 2004, 02:57:51 PM »

Offline excalibur

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Advantages of citrate buffer?
« Reply #1 on: October 05, 2004, 03:41:52 PM »
There really is no advantage to using citrate buffer vs any other antigen retieval solution. The main thing is the heating action breaks your aldehyde bonds to uncover your antigen. I use citrate buffer simply by choice and have never had a problem with it.

I place my slides in the buffer in a plastic slide holder. I then fill the bottom of a Black & Decker rice steamer with distilled water. Place the large steaming basket in the steamer. Put the coplin jar in the basket and then the lid to the steamer on top of that. I steam for 1 hour, let the entire thing cool for 25 minutes, then slowly rinse with distilled water.

Start IHC protocol as usual. Good luck!

Paula
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline Tony

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Advantages of citrate buffer?
« Reply #2 on: October 05, 2004, 03:52:14 PM »
At one time, I even tried tap water and distilled water to replace citrate buffer and it worked although it was not as good as citrate buffer. So I think pH and heating temp and time are more important than type of buffers.

Tony

Offline ole

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Advantages of citrate buffer?
« Reply #3 on: October 06, 2004, 07:35:18 AM »
Hi

Some years ago by mistake i used TBS instead of citratebuffer as a retrieval buffer (they are next to eachother in the frigde). It was not many different ag so of course i cant give a conclusion. At least at that time on the few antigens i detected i got about the same result with TBS as i would with citrate buffer.
i replied on a similar question some time ago (link below)

http://www.ihcworld.com/forum/viewtopic.php?t=569

Ole

Offline REmerson

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advantages of citrate buffer
« Reply #4 on: December 02, 2004, 10:37:51 AM »
The pH is the important part.  Citrate buffer at pH6.0 is more effective than TBS at pH6.0.  I am not exactly sure of the chemistry behind it, but I have run parallel experiments with both buffers and citrate worked noticeably better.

Offline REmerson

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pardon me
« Reply #5 on: December 02, 2004, 10:54:09 AM »
Pardon me.....I meant Tris pH 6.0, not TBS.
I think it is a combination of pH and salt.

Offline lldewe

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Tris-EDTA/EGTA pH 9 as the heating buffer
« Reply #6 on: December 14, 2004, 07:28:35 PM »
This protocol indicates that this is a better HEIR buffer than citrate buffer.

http://www.nordiqc.org/Run-11/Assessment/assessment-CD31.htm

What is a protocol for making the Tris-EDTA/EGTA buffer?

Thanks!
Lorie

Offline ole

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Advantages of citrate buffer?
« Reply #7 on: December 15, 2004, 04:35:36 PM »
Hi Lorie

This "protocol" indicates that TE pH 9,0 might be better than citrate buffer, and/or its easier to get optimal staining using TE-buffer when detecting CD31.  I find TE-buffer pH9,0 to be the best AR-buffer for the majority of the antigens we are staining,
but other buffers might work better than TE for other antigens. It depends on how you do it, the HIER-technique. I know some does not get the TE buffer to work well at all.

Tris EDTA buffer pH 9,0

1mM EDTA
10mM Tris
Check pH. (Usually you dont need to adjust pH).

eg
To 1litre aq dest, add 1,21g Tris and 0,4g EDTA.

You can store it many weeks or months in frigde, or about 1 week in RT. I have encountered that TE buffer stored for many weeks? (i cant remember how many days exactly) in RT does not perform as well as one made up fresh.

Ole

Offline lldewe

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Thanks
« Reply #8 on: December 16, 2004, 12:26:57 PM »
I'm going to give it a try today!

Lorie

Thanks
« Reply #8 on: December 16, 2004, 12:26:57 PM »